The goal is to identify mechanisms that induce and regulate growth in a normally nonproliferating, highly differentiated tissue. Rat liver, a well studied model whose cells are normally in a state of growth arrest (Go phase), undergoes a remarkable burst of proliferative activity in response to an excessive metabolic workload, imposed by the body, and set in motion by partial hepatectomy. The aim is to find molecular mechanisms through which this workload is translated into liver growth. The project is pertinent to restoration or repair of damaged tissues, to further development of an artificial liver, and to our understanding of neoplasia. The working hypothesis is that a large metabolic imbalance elicits hormonal or chemical signals through which the body communicates its needs. These generate a two step response: (a) non specific stress moves the cells into early G1 ("priming", which is reversible), and (b) synergistic combinations of the signals and locally produced short range peptide growth factors mediate progression through G1 to S phase. We are exploring ways in which these growth effectors alter the expression of selected members of the IEGR (immediate early growth response) and C/EBP gene families. This involves extensive use of primary hepatocyte cultures, and parallel studies in animal models to insure physiological relevance. Activation of the IEGR genes indicates that the cells are primed, and decreased expression of C/EBPalpha indicates progression through G1. Conversely, in G-O, IEGR genes are inactive and C/EBPalpha is abundantly expressed. C/EBPalpha regulates metabolic genes and suppress growth, a dual role useful for assessment of our hypothesis, which is supported by our finding that EGF augments growth associated changes in its expression that are counteracted by TGFbeta, a potent growth inhibitor. We propose: l) To explore how other hormone/growth factor combinations compare with our usual insulin/EGF combination in terms of DNA synthesis and activation of the genes mentioned (immunostaining, Northern and Western blots, gel mobility shifts); 2) To continue to explore alternative systems, especially hepatocyte "spheroids"; 3) To investigate the priming and progression stages separately, both in vitro and in vivo, utilizing a noninvasive metabolism-induced hepatocyte growth activation model. In all instances DNA synthesis and gene activities will be compared to normal and regenerating liver. These new approaches should allow basic physiological and molecular biological observations to be integrated into a more comprehensive formulation of how liver regeneration operates.